The present invention relates generally to protein chemistry, and specifically to processes for purifying reoombinant proteins produced by high level expression in microorganisms.
Interleukin-1 (IL-1) is a lymphokine released by macrophages in response to immunogenic stimulation. This polypeptide has been associated with a complex spectrum of biological activities. IL-1 is a primary immunostimulatory signal capable of inducing thymocyte proliferation via induction of interleukin-2 release, and stimulating proliferation and maturation of B-lymphocytes. In addition, IL-1 has been linked with prostaglandin production and induction of fever, and with promotion of wound healing. Reviews of the literature relating to IL-1 include Oppenheim et al., Immunol. Today 7:45 (1986), and Durum et al., Ann. Rev. Immunol. 3:263 (1985).
Human IL-1 activity resides in two distantly related proteins, herein designated IL-1.alpha. and IL-1.beta.(March et al., Nature 315:641 (1985)). Both molecules are normally synthesized as larger precursors having molecular weights of about 30,000 daltons, which are subsequently proteolytically processed to yield mature forms having molecular weights of approximately 17,500 daltons.
Recently, cDNAs coding for both human IL-1 species have been cloned and expressed in microorganisms. This achievement should enable production of sufficient quantities of IL-1.alpha. and IL-1.beta. to permit therapeutic use. However, difficulties have been encountered in purification of active, nondenatured IL-1 species from cells capable of high level expression of the recombinant proteins. These difficulties have been attributed to the observation that the recombinant protein appears to be concentrated by the producing organisms in an insoluble form.
Gubler et al., J. Immun. 36:2492 (1986) reported a process for crude purification of IL-1.alpha. expressed in E. coli. However, this process involved use of the denaturing agents guanidine hydrochloride and urea to enhance solubilization of the recombinant proteins.
The present invention provides a rapid, efficient acid-mediated recombinant protein solubilization process for isolating recombinant IL-1 from microbial cell cultures.